The adverse effects of reactive intermediates derived from toxic chemicals result primarily from covalent binding to macromolecules and lipid peroxidation. Because of the central role of lipid peroxidation in the mechanisms of toxicity, many investigations of this process have been made. Fatty acid hydroperoxides are formed not only in free radical- mediated toxicities, but also physiologically as products of the peroxidation of polyunsaturated fatty acids (PUFA) by prostaglandin H synthases and lipoxygenases. One of the major reactions of PUFA-derived hydroperoxides is with hematin and various hemoproteins. Although the formation of PUFA-derived free radicals was proposed by Tappel in 1953 and free radicals were subsequently detected with ESR, the mechanism(s) of their reaction with hemoproteins has been the subject of considerable debate. The different mechanisms proposed for the reaction of hemoproteins with hydroperoxides dictate that the initial radical produced is either the peroxyl radical (as predicted by the heterolytic peroxidase mechanism), the alkoxyl radical (via the homolytic mechanism), or both the peroxyl and alkoxyl radicals (via the Haber-Weiss-type mechanism). The reactions of the initial radical that occur subsequent to its production can be suppressed by increasing the concentration of the spin trap used in the detection and identification of these reactive species. Thus, as more spin trap is added, more of the initial radical is trapped. This prevents the initial radical from undergoing reactions with other species or itself that lead to secondary radicals. By doing this, one can determine which radical is the primary or initial radical in a multiple species ESR spectrum. We used this strategy to determine whether the peroxyl or the alkoxyl radical was the initial radical produced by the reaction of hemoproteins with hydroperoxides. In every case examined thus far (i.e., cytochrome c, hematin, and cytochrome P- 450) the alkoxyl radical adduct completely dominated at the highest DMPO concentrations. Thus, the ESR data provides strong evidence for the homolytic scission of the hydroperoxide O-O bond by hemoproteins initially producing alkoxyl radicals.